Fibrinogen is a soluble plasma protein consisting of two pairs of three protein chains designated A.alpha., B.beta., and .gamma. to give a configuration of (A.alpha.B.beta..gamma.).sub.2 for the intact fibrinogen molecule. The A.alpha., B.beta., and .gamma. chains interact by a series of 29 disulfide bonds among the six protein chains. Fibrinogen is converted to insoluble fibrin by the action of thrombin and Factor XIIIa. Thrombin cleaves four short fibrinopeptides from fibrinogen: two fibrinopeptide A (FpA) fragments and two fibrinopeptide B (FpB) fragments from the amino termini of the A.alpha. and B.beta. chains, respectively. The resulting six chain proteins are monomers, which assemble to form a fibrin matrix; this fibrin matrix is the principal component of blood clots.
Bacterial systems have been used to express the individual recombinant fibrinogen chains as monomers. Bolyard and Lord, Blood, 73, 1202 (1989); Bolyard and Lord, Gene 66, 183 (1988); Lord, DNA, 4, 33 (1985). A significant drawback of bacterial expression systems is that they cannot produce intact, biologically active fibrinogen molecules. Intact human fibrinogen has been expressed in yeast. Roy et al., J. Biol. Chem. 270, 23761 (1995).
Farrell et al., Biochemistry 30, 9414 (1991) disclose a method of producing recombinant human fibrinogen in BHK (baby hamster kidney) and HepG2 human liver cells. These investigators employed the BHK and HepG2 systems to study post-translational modification of fibrinogen variants. Farrell et al. also disclose a method of purifying recombinant fibrinogen from conditioned BHK and HepG2 culture media by immunoprecipitation with protein A-SEPHAROSE.RTM.. The immunopurified material was used for analytical purposes in clotting assays and chromatographic and electrophoretic analysis. Likewise, Roy et al., J. Biol. Chem. 266, 4758 (1991) purified recombinant human fibrinogen from COS-1 cells and Hartwig and Danishefsky, J. Biol. Chem. 266, 6578 (1991) purified recombinant human fibrinogen from COS-1, HepG2 and HepG3 cells by immunoprecipitation for analytical characterization.
Binnie et al., Biochemistry 32, 107 (1993), disclose a method of producing recombinant human fibrinogen in cultures of Chinese Hamster Ovary (CHO) cells using a two-step transfection procedure. First, CHO cells are cotransfected with expression vectors encoding the A.alpha. and .gamma. chains of human fibrinogen. Cell lines derived from clones expressing high levels of both the A.alpha. and .gamma. chains are then cotransfected with a B.beta. chain expression vector to give cell lines expressing all three recombinant human fibrinogen chains. The recombinant A.alpha., B.beta., and .gamma. chains assemble to form intact human fibrinogen. The recombinant human fibrinogen is purified from the conditioned culture medium by protamine SEPHAROSE.RTM. chromatography. One disadvantage of the method of Binnie et al. is that the recombinant fibrinogen is present at low concentrations in large volumes of conditioned culture medium. Both the yield and purity of the purified fibrinogen are adversely affected under these conditions.
Farrell et al., J. Biol. Chem. 269, 226 (1994), disclose a method of purifying recombinant human fibrinogen from cultures of BHK cells by two sequential rounds of affinity chromatography. First, the conditioned culture medium is passed through a protamine-agarose column. The fibrinogen in the eluate is further purified over a column bearing immobilized peptide corresponding to the carboxyl terminus of the fibrinogen .gamma. chain. This method also results in large volumes of conditioned culture medium containing only low concentrations of fibrinogen; Farrell et al. state that the conditioned culture medium was applied to the protamine-agarose column at 30-40 ml/hour over a five-day period. Id. at page 227.
Accordingly, there remains a need in the art for improved methods of producing and purifying recombinant fibrinogen from long-term mammalian cell cultures.